Determination of Transition Metals in Serum and Whole Blood by Ion Chromatography

Importance of the Topic

Determining trace transition metals in serum and whole blood plays a vital role in clinical chemistry. Abnormal levels of copper, zinc, iron and other metals are linked to diseases such as Wilson’s disease, anemia and various malignancies. Reliable analysis supports diagnosis, therapy monitoring and nutritional assessment.

Objectives and Study Overview

This study presents an ion chromatography (IC) approach for quantifying transition metals in physiological fluids. The goal is to offer a sensitive, precise alternative to atomic absorption spectrophotometry (AAS), with simplified sample preparation and the ability to speciate metal oxidation states.

Methodology and Used Instrumentation

Two IC separations are described using a mixed anion/cation‐exchange column (IonPac CS5A) and a guard column (CG5A). Two eluents are employed:

• PDCA eluent: pyridine-2,6-dicarboxylic acid complexing for Fe(II/III), Cu, Ni, Zn, Co, Cd and Mn.
• Oxalic acid eluent: moderate complexing for Pb, Cu, Co, Zn, Ni (Cd and Mn coelute).

After chromatographic separation, metals react postcolumn with 4-(2-pyridylazo)resorcinol (PAR) and are detected by absorbance at 520–530 nm.

Recommended equipment (Dionex DX-500 system):

• GP40 Gradient Pump
• AD20 Absorbance Detector
• LC20 Chromatography Module
• PC10 Postcolumn Pneumatic Controller
• PeakNet Chromatography Workstation

Reagents include MetPac PDCA and Oxalic Acid Eluent concentrates, PAR postcolumn reagent, trace-metal grade acids and hydrogen peroxide.

Sample preparation involves protein removal and acid digestion. Serum and whole blood are treated with nitric and sulfuric acid plus hydrogen peroxide, evaporated, then diluted in eluent. Alternatively, trichloroacetic acid deproteinization can be used for serum/plasma.

Main Results and Discussion

Using the PDCA eluent, Fe(II) and Fe(III) species are resolved along with Cu, Ni, Zn, Co, Cd and Mn. Oxalate eluent separates Pb, Cu, Co, Zn and Ni. Tables of precision and recovery demonstrate:

• Whole blood: recoveries ~95–100% for Zn, Fe(III) RSD ~2.7%
• Serum: Cu and Zn recoveries ~95–98%, MDLs of 45–70 µg/L

Oxygen removal by sodium sulfite degassing is critical to prevent oxidation of Fe(II). All metal components in the flow path must be avoided to reduce contamination. PAR reagent is sensitive to oxidation and must be stored under inert gas.

Benefits and Practical Applications

This IC method offers:

• High sensitivity and specificity in complex matrices
• Low limits of detection comparable to AAS
• Rapid, reproducible separations
• Ability to speciate iron oxidation states
• Minimal sample preparation and reduced matrix interferences

It is suitable for clinical diagnostic laboratories, nutritional studies and toxicology screening.

Future Trends and Potential Uses

Advances may include coupling IC with mass spectrometry for multi‐element analysis, microcolumn technology to reduce reagent consumption and automated sample preparation. Expanded panels could integrate other trace and rare earth metals for comprehensive metallomic profiling.

Conclusion

Ion chromatography with postcolumn PAR detection provides a robust and efficient method for transition metal analysis in serum and whole blood. It addresses limitations of AAS, enabling precise speciation and high throughput in clinical and research settings.

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